Subtypes of Macrophages and Interaction of Macrophages with the RPE. In collaboration with Dr. Siamon Gordon in Oxford and Dr. Miller at the NEI, we have initiated studies on human macrophages focusing on 1) the interaction of human macrophages with human retinal pigmented epithelial cells and 2) phenotypic and functional heterogeneity of human macrophages and its implication in autoimmune intraocular inflammatory diseases. We have found that human RPE cells can interfere with the normal processes of differentiation and maturation of macrophages. We then evaluated monocytes' role in the development of intermediate, posterior and panuveitis. Preliminary data showed that immuno-suppression enrich anti-proinfllammatory monocytes in patients' peripheral blood, implying that regulatory monocytes can serve as a prognosis marker for uveitis. We further showed that different types of monocytes interact T cells in different modes. Immune factors leading to CNV By adapting and modifying a commonly used laser induced CNV mouse model, we have established a strategy to investigate the involvement and potential mechanisms of immune responses in CNV, the most common complication in AMD and other important eye diseases. We have combined flow cytometry, vibrotome immunohistochemistry and flat-mount techniques plus confocal immunomicroscopy to evaluate the model. We have discovered that there is a dynamic and complex movement of immune cells during the development ofCNV in the mouse. Immune cells that are involved in CNV include neutrophils, monocytes, macrophages, NK cells and microglial cells. There is an apparent differential influx of immune cells in that neutrophils and NK cells move in very early followed by macrophages and microglial cells. The macrophages and microglial cells are present as long as 2-months after laser induced CNV. In addition, by using gene knock-out mice and therapeutic intervention using defined biological reagents, we have found that T cells and B cells are probably not important for the early development of CNV in this mouse model. However, the scavenger receptors, for example, SRA and MARCO, play roles in CNV formation. We have observed that SRA and MARCO knock out mice developed smaller-size CNV leisions. Less neutrophil/macrophage recruitment appears to be involved in this process. Complement and the immune response. Several reports have associated SNPs of complement factor H with either susceptibility to or protection against AMD. This association has been attributed to involvement of the innate immune system, presumably through macrophage activity and Toll-like receptors. We have looked at this question differently; rather that complement and its components may be part of the acquired immune system. We have showed that C5a promoted IL-22 and IL-17 expression from human CD4+ T cells of AMD patients and normals accompanied by the expression of the transcription factor BATF. This effect was dependent on B7, IL-1beta and IL-6 expression from monocytes. Intriguingly, we found significantly increased levels of IL-22 in the serum of 22 AMD patients and the vitreous of one AMD patient as compared to non-AMD controls, strongly suggesting possible roles of IL-22 and IL-17 in the inflammation that contributes to AMD. We also showed that IL-22 directly decreased retinal pigment epithelium (RPE) viability. RPE loss is a consistent feature of the development of advanced AMD. In addition to the role of C5a in T cell differentiation, C5a also decreases human RPE cell viability and abrogates the suppressive effect of RPE on T cells. Thus, our data suggest an underlying mechanism of chronic inflammation leading to retinal degeneration which may herald a new approach to treat AMD.